Reading, detection or quantification method, hybrids or complexes used in said method and the biochip using same

ABSTRACT

The invention relates to a method of reading, detecting or quantifying at least one biological reaction, on a support, between either a recognition molecule and a labeled target molecule or between a target molecule and a labeled detection molecule. The inventive method comprises treating the support under physicochemical conditions that allow the following: either the separation of the recognition molecule and the labeled target molecule or the separation of the target molecule and the labeled detection molecule. The inventive method further comprises producing images before and after the physicochemical treatment that can be used to determine the specific and non-specific bindings between the different molecules. The invention also relates to hybrids and complexes used in the inventive method and to a biochip containing the same which is used to carry out the inventive method. The invention is particularly suitable for use in the field of diagnosis.

The present invention relates to a method of reading, a method ofdetecting and a method of quantifying chemical or biological reactionsperformed on a support, which support may consist of a Petri dish, amicrotiter plate, a biochip and the like. The present invention alsorelates to a support comprising a plurality of, that is to say at leasttwo, zones of molecular recognition, each recognition zone containing atleast one recognition molecule. While there are at least two recognitionmolecules per recognition zone, all the recognition molecules arestructurally identical. The present invention finally relates to hybridsor complexes which may be used on such supports.

The expression biochip is understood to mean a chip having at itssurface a plurality of recognition zones, that is to say at least onehundred recognition zones, endowed with molecules having recognitionproperties. In the remainder of the text, and by a misuse ofterminology, the term biochip is used independently of whether the chipis intended for chemical or biological analysis. The concept of biochip,more precisely of DNA chip, dates from the beginning of the 1990s.Nowadays, this concept has been extended since protein chips havestarted to be developed. It is based on a multidisciplinary technologyincorporating microelectronics, nucleic acid chemistry, image analysisand computing. The principle of operation is based on a molecularbiology foundation: the phenomenon of hybridization, that is to say thepairing by base complementarity of two DNA and/or RNA sequences.

The biochip method is based on the use of probes (DNA sequencesrepresenting a portion of a gene or an oligonucleotide), attached to asolid support on which a sample of nucleic acids labeled directly orindirectly with fluorochromes is caused to act.

However, it is quite possible to use other more conventional supportssuch as Petri dishes, microtiter plates which contain a number ofseparate wells, and the like. The expression support is understood tomean an analytical surface which contains only a few recognition zones,generally at most one hundred recognition zones. Each recognition zonecomprises at least one molecule having recognition properties

In all cases, the probes, also called recognition molecules, arepositioned in a specific manner on the support or chip and eachhybridization gives information on each gene represented. Thisinformation is cumulative, and makes it possible to detect the presenceof a gene or to quantify the level of expression of this gene in thetissue studied. After hybridization, the support or chip is washed, readfor example by a scanner and the analysis of the fluorescence isprocessed by a computer.

The support or chip which serves to attach the probes generally consistsof a flat or porous surface composed of materials, such as:

-   -   glass, an inexpensive, inert and mechanically stable material;        the surface may be covered with a Teflon screen which delimits        hydrophilic and hydrophobic zones,    -   polymers,    -   silicon, and    -   metals, in particular gold and platinum.

However, it is also possible to use particles, for example magneticparticles, as described in patent applications WO-A-97/34909,WO-A-97/45202, WO-A-98/47000 and WO-A-99/35500 from one of theapplicants.

To attach the probes (or recognition molecules), three main types ofmanufacture are distinguishable.

There is first of all a first technique which consists in depositingpresynthesized probes. The attachment of the probes occurs by directtransfer, by means of micropipettes, microtips or by an ink-jet typedevice. This technique allows the attachment of probes having a sizeranging from a few bases (5 to 10) up to relatively large sizes of 60bases (imprinting) to a few hundreds of bases (microdeposition):

-   -   Imprinting is an adaptation of the method used by ink-jet        printers. It is based on the propelling of very small spheres of        fluid (volume<1 nl) and at a rhythm which may be up to 4000        drops/second. Imprinting does not involve any contact between        the system releasing the fluid and the surface on which it is        deposited.    -   Microdeposition consists in attaching probes which are a few        tenths to a few hundredths of bases long to the surface of a        glass slide. These probes are generally extracted from databases        and exist in the form of amplified and purified products. This        technique makes it possible to prepare chips called microarrays        carrying about ten thousand spots, called recognition zones, of        DNA on a surface of slightly less than 4 cm². There should        however not be forgotten the use of Nylon membranes, called        “macroarrays”, which carry products which have been amplified,        generally by PCR, with a diameter of 0.5 to 1 mm and the maximum        density of which is 25 spots/cm². This highly flexible technique        is used by many laboratories. In the present invention, the        latter technique is considered as forming part of biochips. It        is possible however to deposit, at the bottom of a microtiter        plate, a certain volume of sample into each well, as is the case        in patent applications WO-A-00/71750 under priority of 20 May        and of 6 Dec. 1999 and FR00/14896 of 17 Nov. 2000 from one of        the applicants, or to deposit, at the bottom of the same Petri        dish, a certain number of drops separated from each other,        according to another patent application FR00/14691 of 15 Nov.        2000 from this same applicant.

The second technique for attaching probes to the support or chip iscalled in situ synthesis. This technique results in the production ofshort probes directly at the surface of the chip. It is based on thesynthesis of oligonucleotides in situ, invented by Edwin Southern, andis based on the method of oligonucleotide synthesizers. It consists inmoving a reaction chamber, where the oligonucleotide extension reactionis taking place, along the surface of glass.

Finally, the third technique is called photolithography, which is amethod at the origin of the biochips developed by Affymetrix. It alsoinvolves an in situ synthesis. Photolithography is derived frommicroprocessor techniques. The surface of the chip is modified by theattachment of photolabile chemical groups which can be activated bylight. Once illuminated, these groups are capable of reacting with the3′ end of an oligonucleotide. By protecting this surface with masks ofdefined shapes, it is possible to illuminate and therefore activateselectively zones of the chip where it is desired to attach either ofthe four nucleotides. The successive use of different masks makes itpossible to alternate protection/reaction cycles and therefore toproduce the oligonucleotide probes on spots of about a few tenths of asquare micrometer (μm²). This resolution makes it possible to create upto several hundreds of thousands of spots on a surface of a few squarecentimeters (cm²). Photolithography has advantages: massively parallel,it makes it possible to create a chip of N mers in only 4×N cycles.

All these techniques can of course be used with the present invention.

Methods using such supports or biochips can be essentially used for:

-   -   searching for the presence or absence of a pathogenic agent, for        example for a bacterium in meat,    -   searching for the presence or absence of mutations. Knowing the        molecular structure of the gene, oligonucleotides are        manufactured which represent all or the complementary part of        this gene. In the presence of the biological sample,        hybridization will occur between all or part of said gene, which        is generally labeled, for example by fluorescence, and the        oligonucleotides, and the image obtained by fluorescence makes        it possible to know if there is a mutation and in which position        it is situated. In this application, the use of DNA chips is        equivalent to sequencing for a diagnosis of mutation, with a        huge advantage in terms of speed, and    -   measuring the level of expression of genes in a tissue. The chip        network carries a very large number of probes which correspond        to all the genes of the species to be studied. A sample, for        example of previously amplified mRNAs, which represents the        active genes of the tissue, is hybridized. Fluorescence analysis        makes it possible to know the level of expression of each gene.

The efficiency of such supports and biochips was tested on well knownbiological systems such as yeast (cell cycle, respiratory metabolism,fermentation and the like). Comparison of the results obtained by thechips with those previously obtained by other approaches showedagreement for the genes whose expression was already well known in thesebiological systems. This work has thus made it possible to validate thetechnology of biochips in relation to the more conventional supportswhich we have mentioned.

Moreover, companies are involved in novel methods which also allowgenomic analyses to be performed in parallel. Thus, the microbeadtechnique makes it possible to attach probes to microspheres carrying anindividual “tag” or mark, and more precisely a genetic code. Afterreaction, the reading of this mark makes it possible to unambiguouslyidentify a microbead and therefore the probe present at its surface. Themicrospheres are brought into contact with the test sample labeled withone or more fluorochromes. The mixture is then analyzed by flowcytometry which will, on the one hand, identify each bead according toits “tag”, and, on the other hand, measure the fluorescence indicatingan effective hybridization reaction.

DNA chips have paved the way to a novel instrumentation in molecularbiology which can even incorporate various stages of analyses inminiaturized form, see in this regard patent applications WO-A-00/78452under priority of 22 Jun. 1999 and FR00/10978 filed on 28 Aug. 2000 fromone of the applicants. Furthermore, as already indicated above, the veryhigh interest recently created by proteomics, the key post-genomicsdiscipline, is accompanied by the emergence of the concept of proteinchips. These also form part of the supports according to the invention.

The recognition molecules may be, for example, oligonucleotides,polynucleotides, proteins such as antibodies or peptides, lectins or anyother ligand-receptor type system. In particular, the recognitionmolecules may contain DNA or RNA fragments.

When the support is brought into contact with a sample to be analyzed,the recognition molecules are capable of interacting, for example byhybridization in the case where they are nucleic acids or by formationof a complex in the case where they are antibodies or antigens, withtarget molecules present in a liquid biological sample.

Thus, by equipping a biochip with a plurality of recognition zones withvarious different recognition molecules, where each recognition moleculeis specific for a target molecule, it is possible to detect and possiblyquantify a large variety of molecules contained in the sample. It is ofcourse obvious that each recognition zone contains only one type ofrecognition molecules which are identical to each other.

The support-recognition molecule-target molecule assembly can bedetected by a detection molecule. The support-recognitionmolecule-target molecule-detection molecule assembly constitutes a testin a sandwich format. Tests in a sandwich format are widely used indiagnosis, whether in molecular diagnosis, ELOSA (Enzyme-LinkedOligo-Sorbent Assay) test for example, or in immunological diagnosis,for example ELISA (Enzyme-Linked Immuno-Sorbent Assay) test for example.In general, they comprise a recognition molecule, such as a nucleic acidprobe or an antigen (case of an antigen sandwich) or an antibody (caseof an antibody sandwich), which serves to capture a target, which willconsist respectively of a nucleic acid probe or an antibody or anantigen. This recognition molecule is attached to a solid support in amanner known to a person skilled in the art, for example:

-   -   either by adsorption,    -   or by direct coupling,    -   or via an intermediate protein, such as for example avidin or        protein A,    -   or via polymers.

The recognition molecule and target molecule assembly is then detectedby a detection molecule, which will respectively consist of a nucleicacid probe or an antibody or an antigen. This detection molecule carriesor may be subsequently combined with a marker, which marker is necessaryin order to allow detection and/or quantification. The detectionmolecule, whether still combined with a marker or not, will still becalled detection molecule.

In the text which follows, the term “hybridization” will be associatedwith the attachment of a nucleic acid to another nucleic acid, whereasthe term “complexing” will be associated with the attachment of anantibody to an antigen. On the other hand, the term “attachment” willhave a broader definition which may relate at the same time to:

-   -   the attachment of a nucleic acid to another nucleic acid,    -   the attachment of an antibody to an antigen, or    -   the attachment of a biological molecule to a support.

The tests currently available are tests such as those developed by oneof the applicants for immunological assays or the DNA chips developed bythe company Affymetrix (“Accessing Genetic Information with High-DensityDNA arrays”, M. Shee et al., Science, 274, 610-614. “Light-generatedoligonucleotide arrays for rapide DNA sequence analysis”, A. CavianiPease et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 5022-5026), formedical diagnosis. In this technology, the capture probes are generallyof small sizes, of around twenty nucleotides.

In the ELOSA field, that is to say in the field of detection of nucleicacids, see in this regard the patent application filed by one of theapplicants WO-A-91/19812, there are defined in the same manner a captureoligonucleotide (recognition molecule), a target nucleic acid (targetmolecule) which is either DNA or RNA, and a detection oligonucleotide(detection molecule). Capture and detection oligonucleotides arecomplementary to part of the target but at the level of the regions ofthe target which are respectively structurally and physically different,such that the capture and detection oligonucleotides cannot hybridize toone another.

Whether in molecular diagnosis or in immunological assays, the detectionelements carry a marker which allows the detection and/or thequantification of the target. The expressing labeling is understood tomean the attachment of a marker capable of directly or indirectlygenerating a detectable signal. Various markers have been developed withthe permanent objective of improving sensitivity. They may be eitherradioactive, enzymatic, fluorescent, or, as described more recently, inthe form of nanoparticles. These nanoparticles are different frommicroparticles, in particular in their size which remains considerablyless than a micron. Because they are less trivial, they will be thesubject of a more detailed disclosure later.

-   -   A nonlimiting list of these markers, which makes it possible to        carry out imaging with a single marker, will be described in the        remainder of the description.

Indirect systems may also be used, such as for example ligands capableof reacting with an antiligand. The ligand/antiligand pairs are wellknown to a person skilled in the art, which is the case for example ofthe following pairs:

-   -   biotin/streptavidin,    -   hapten/antibody,    -   antigen/antibody,    -   peptide/antibody,    -   sugar/lectin,    -   polynucleotide/complementary to the polynucleotide,    -   successive histidine sequence, called “tag”, for a metal, for        example nickel.

In this case, it is the ligand which carries the linking agent. Theantiligand may be detectable directly by the markers described in thepreceding paragraph or may itself be detectable by a ligand/antiligand.

These indirect detection systems can lead, under certain conditions, toa signal amplification. This signal amplification technique is wellknown to a person skilled in the art, and reference may be made toprevious patent applications FR98/10084 or WO-A-95/08000 from one of theapplicants or to the article J. Histochem. Cytochem. 45: 481-491, 1997.

Moreover, the applicants have jointly filed a patent applicationPCT/FROO/03359 under French priority of 2 Dec. 1999 on a signalamplification technique. However, the latter does not function on thebasis of the teachings drawn from the preceding documents, whichincrease the number of markers at the level of the sites of attachmentof said markers, but rather uses a particular coating at the surface ofthe support, which coating is based on a thin layer of a material chosenfrom silicon nitride, silicon carbide, titanium oxides, aluminum oxide,ZrO₂, ZrO₄Ti, HfO₂, Y₂O₃, diamond, MgO, oxynitrides (Si_(x)O_(y)N_(z)),fluorinated materials, YF₃, MgF₂. These two techniques may becumulative.

As mentioned above, the markers may also be in the form ofnanoparticles. The first experiments using conjugates with nanoparticlesdate back to 1980. They were motivated by the search for ultrasensitivelabeling techniques which avoid the use of radioactivity or the use ofenzymatic labelings which require not only time but also the use oftoxic reagents. They have been widely developed and used since then andmany types of particle have been developed, such as microparticles,nanoparticles of latex or particles of colloidal gold or alternativelyfluorescent particles. In the text which follows, the term “particles”will be used without distinction for microparticles or nanoparticles orother particles of different sizes. Many particles are now commerciallyavailable (Bangs, Milteny, Molecular Probes, Polyscience, Immunicon).

Labeling experiments using nanoparticles were started in the field ofimmunological assays by J. H. W. Leuvering, P. J. H. M. Thal, M. Van derWaart and A. H. W. M. Schuurs, Sol Particle Immunoassay (SPIA). Journalof Immunoassay 1(1):77-91, 1980.

The use of nanoparticles has allowed access to other technologies suchas atomic force microscopy for reading immunological assays, with whichtechnology, on a TSH detection model, a sandwich system mounted on asilicon support using anti-TSH detection antibody conjugates bound togold nanoparticles has made it possible to obtain a sensitivity of theorder of 1 pM. On this subject, reference may be made to the followingtwo publications:

-   -   Agnès Perrin, Alain Theretz, and Bernard Mandrand Thyroïd        stimulating hormone assays based on the detection of gold        conjugates by scanning force microscopy. Analytical biochemistry        256:200-206, 1998, and    -   Agnès Perrin, Alain Theretz, Véronique Lanet, S. Vialle, and        Bernard Mandrand Immunomagnetic concentration of antigens and        detection based on a scanning force microscopic immunoassay.        Journal of immunological methods 8313, 1999

The use of nanoparticles for the detection of nucleic acids has beenobserved since only very recently. The work carried out in the field ofimmunology finds its equivalent in the field of nucleic acids. There isno fundamental innovation either in the labeling methods or in thedetection methods. Since the ELISA plate no longer exists, it is indeedreplaced by a flat support. The nanoparticles are then detected bymicroscopy, by amplification for surface plasmon resonance, called“biosensor” technique, or by measurement using AFM. Overall, thebiological models remain simple models.

The work by Taton, T. A., Mirkin C. A. and Letsinger R. L. relating to:“Scanometric DNA array detection with nanoparticle probes” Science 2000Sep. 8; 289(5485):1757-60, presents the use of gold nanoparticlescoupled to detection oligonucleotides of 15 bases for detecting a targetof 27 bases which is captured on a probe of 12 bases. The detectionsignal is amplified by labeling with silver. For high targetconcentrations of the order of 10 nanoMolar (nM), the detection of theparticles is carried out by eye by observing the passage of a pinkcolor. For the lowest concentrations, 100 picoMolar (pM) for example,the detection requires the use of a scanner. The sensitivity reached isof the order of 50 femtoMolar (fM). The authors are capable ofdissociating the nanoparticles from the surface by heating, which tendsto show the specificity of their labeling. However, according to theexperiment of the applicants and because of the respective sizes of thedetection oligonucleotides (15 bases), of the target oligonucleotides(27 bases) and of the capture oligonucleotides (12 bases), there mustnecessarily be dissociation between said nanoparticles and the detectionoligonucleotides. This work is based, in addition, on a set ofexperiments which have been the subject of several publications (1996;1997; 1997; 2000) and several patent applications and patents, such asWO-A-98/04740.

These researchers carry out a dissociation of the nanoparticles whichdoes not distinguish the nanoparticles combined with a detectionmolecule with no mismatch with the target and the nanoparticles combinedwith a detection molecule with a mismatch with said target. In the textthat follows, the expression “dissociation” is understood to mean inparticular the separation between the recognition molecule and thetarget molecule and/or the separation of the target molecule and thedetection molecule. If this dissociation is thermal, the physicalcharacteristic involved consists of the melting point (Tm) whichcorresponds to the temperature zone where the DNA or RNA molecules aredenatured. More precisely, this temperature corresponds to a state wherea population of identical oligonucleotides, in the presence of anidentical quantity of complementary oligonucleotides, is 50% indouble-stranded form, that is to say paired, and 50% in single-strandedform.

While labelings with nanoparticles make it possible to obtain goodsensitivity, there is nevertheless a compromise to be made between theincrease in the specific signal and the decrease in the nonspecificsignal. The nonspecific signal is of course limiting for improving thesensitivity and the dynamics of the method. The authors solve theseproblems by optimizing the experimental conditions. As a guide, someauthors (Okano et al.; Anal. Biochem. 202:120-125; 1992) have optimizedthe concentration of detection oligonucleotides per particle, on the onehand, and the concentration of particles in the solution, on the otherhand. They have also succeeded in reducing the nonspecific by adding BSA(bovine serum albumin).

As regards the use of nanoparticles for amplifying a signal obtained ona biosensor, reference is made to the work of Lin He, Michael D. Musick,Scheila R. Nicewaener, Franck G. Salinas, Stephen J. Benkovic, MichaelJ. Natan, and Christine D. Keating. Colloidal Au-enhanced surfaceplasmon resonance for ultrasensitive detection of DNA hybridization. J.Am. Chem. Soc. 122:9071-9077, 2000. Detection oligonucleotides (12bases) conjugated with nanoparticles of colloidal gold are used toamplify the detection of a small sized nucleic acid target (24 mers) bysurface plasmon resonance. They obtained a sensitivity of 10 pM, that isto say a surface density of 8.10⁸ molecules/cm² with an improvement insignal intensity of a factor of 100000. They verified the hybridizationspecificity by dissociating the nanoparticles from the surface either byheating or by digesting with restriction enzymes if the restriction siteis present on the hybridized sequence. In this case, the labeling isused to amplify the signal, and the dissociation serves to show that thelabeling results from a specific hybridization.

It is a verification of hybridization. Either the nanoparticles areseparated by heating, in which case the scenario described by Taton(2000) exists and there is no specificity in the removal of the marker,or the nanoparticles are separated by restriction enzymes, in this casethe nanoparticles are removed, for example by washing, while remainingcombined with all or part of the detection molecule and/or the hybrid.There is an oriented character of this separation, but withoutdissociation or with a partial dissociation of the double strands.

Among the state of the art methods using nanoparticles, the document byKubitschko S., Spinke J., Bruckner T., Pohl S. and Oranth N.“Sensitivity enhancement of optical immunosensors with nanoparticles.”Anal. Biochem. 253(1):112-122 from 1997 evokes, in immunologicaldiagnosis, the dissociation of the antibody conjugates in relation tothe antigens. This dissociation is achieved with formic acid whichserves to regenerate a microcomponent which still contains theantibodies for subsequent uses of the microcomponent.

It is more a method of cleaning a microcomponent; as a result, no defacto discrimination is made when the dissociation of the antibodyconjugates is carried out in relation to the antigens which may becombined with the nanoparticles.

U.S. Pat. No. 6,093,370 provides another method which has the objectiveof recovering a DNA captured on a DNA chip. It is a photothermaldissociation carried out with an infrared (IR) laser at 1053 nanometers(nm) which irradiates with a power of between 10 and 100 mW the regionof the chip on which it is desired to recover the DNA. The DNAredissolved in solution is amplified by PCR. The authors show that theyare capable of extracting at least one DNA molecule per 169 nm², that is10-17 mol/1000 μm².

However, they do not use a physicochemical dissociation for determiningthe hybridizations which are real because they are specific (truepositives) from the other hybridizations.

In addition to the dissociation techniques mentioned above, that is tosay by thermal denaturation (heating), by photothermal denaturation (IRlaser), by digestion (restriction enzymes), by chemistry (formic acid),other methods of dissociating nucleic acids exist in the state of theart.

Thus, it is possible to modify the ionic strength of the hybridizationbuffer in order to dissociate the detection oligonucleotide from thetarget. Indeed, the melting point of the oligonucleotides variesaccording to the ionic strength of the buffer. The lower the ionicstrength of the buffer, the less stable the oligonucleotide. It is alsopossible to combine the action of temperature with the bufferconditions.

It is also possible to use PNA (Peptide Nucleic Acid) or any othermolecules used in the state of the art for capturing a nucleic acid. ThePNA/oligonucleotide hybrids have the same thermal stability regardlessof the ionic strength, that is to say that their melting point does notvary according to the ionic strength of the hybridization buffer. If thecapture probe is a PNA and the detection probe is an oligonucleotide, onusing a low ionic strength, the detection probe dissociates from thetarget whereas the target remains hybridized with the capture probe(PNA).

It is possible to chemically modify the detection probes in order tocarry out the dissociation of the DNA under certain conditions. Thesedissociation methods do not relate to the particles which interact withthe surface via the surface properties of the nanoparticles and of thesupport.

It is also possible to modify a base of the oligonucleotide, asdescribed for example by Dreyer et al. (Proc. Natl. Acad. Sci., 82:968-972; 1985). An EDTA group is coupled to a thymidine. In the presenceof DTT, Fe(II) and O₂, a cleavage of the DNA occurs at the level of thethymidine carrying the EDTA.

As it is possible to couple a biotin to the detection oligonucleotide,for subsequent attachment to an avidin, it is also possible to couple achemical group of the homobifunctional or heterobifunctional type(chemical functional groups necessary for its attachment to theoligonucleotide and to the support) which contains an additionalchemical functional group which may be cleaved under certain conditions:for example a photocleavable group or alternatively a group reduced byDTT. In the case of a photocleavable group, exposing the oligonucleotideto a given wavelength cleaves the functional group and dissociates theoligonucleotide from its support.

Another method may also consist in adding a known sequence having asmall size, called “tag”, which has the property of forming a chelatewith a chelating group present on the nanoparticle, via a metal ion. Thetag may be a Histidine tag or any other molecule described by the stateof the art. The chelating group may be an NTA (nitrilotriacetic acid) orany other group described by the state of the art. The dissociation willconsist in using the methods described by the state of the art fordissociating the tag-metal-chelating group interaction (for example useof EDTA).

The dissociation may also be performed by displacing the detectionprobe, present on the nanoparticle, by oligonucleotides having identicalsequences. These oligonucleotides may have either a shorter sequence orthe same sequence with an additional sequence complementary to thetarget. Also, it is possible to use nucleic acid analogs for thisdisplacement by competition, for example PNAs (Peptide Nucleic Acids) oranother analog having the advantage of a neutral backbone.

As regards the dissociation of protein molecules such as antigens and/orantibodies, it is possible to apply any of the methods described in thestate of the art for dissociating the interactions between an antigenand an antibody (numerous techniques known in the field of affinitychromatography). By way of example, it is thus possible to carry out thedissociation by applying acid solutions such as a glycine buffer atacidic pH or solutions with high ionic strength such as 5M LiCl(Kubitschko et al., Anal. Biochem. 253(1):112-122; 1997). It is alsopossible to use denaturing agents such as guanidium chloride, urea oralternatively solutions containing detergents; or methods for digestingproteins with proteases or even endoproteases specific for the antigenor the antibody or alternatively nonspecific for the antigens or theantibodies.

In the case of a double labeling, the dissociation should be oriented,without being denaturing for the proteins so as to allow a secondlabeling. This may be carried out for example by displacement by the useof synthetic peptides, whose sequence corresponds to the sequence of theepitopes of monoclonal antibodies but with a higher affinity.

It is also possible to modify, chemically or by genetic engineeringtechniques, antigens and antibodies in order to carry out thedissociation under certain conditions. These methods do not relate,however, to the protein molecules which interact directly with thesurface in a nonspecific manner. Thus, the modification may relate toone of the proteins (antigen or antibody) by inserting, for example bygenetic engineering, a sequence for cleavage by an endoprotease. In thepresence of the enzyme, there is cleavage of the protein to be detected.The second labeling is performed using a second monoclonal antibodyspecific for the remaining protein sequence. The modification may relateto the end of the protein by adding, for example by genetic engineering,a tag. This tag may be recognized by monoclonal antibodies, thedissociation is then made by displacement using competitor peptides orantibodies. As a guide, the tag added may be a “poly-Histidine”sequence, may contain a sequence for cleavage by endoproteases whichdoes not exist in the sequence, the dissociation is then carried out byenzymatic cleavage. Other sequences such as protein “splice” sequencesmay also be integrated into the protein. The cleavage is made in thepresence of DTT for example.

Finally, it is also possible to add to the detection protein a nucleicacid sequence. A tag is then obtained, as above, which can be recognizedby a nucleic probe which is itself conjugated with a nanoparticle. Thedissociation, oriented or otherwise, can then be carried out by one ofthe techniques described for nucleic acids, provided that the proteinassembly is not denatured in the case of an oriented dissociation.Competition methods are advantageous from this point of view.

All these elements mentioned above are capable of being incorporatedinto the invention in order to further improve the performance thereof.

According to patent application WO-A-99/65926, from one of theapplicants, it is also possible to dispense with the detection moleculeproper in the case of nucleic acids. Each target can be cleavedchemically, enzymatically or physically, while undergoing simultaneouslabeling or otherwise. The presence of a detection molecule is then nolonger necessary since the target molecule is labeled, see in particularpatent application PCT/FR99/01469.

Devices exist for reading molecules which are labeled or otherwise, andwhich may be present in the recognition zones of the chip.

The reading of the recognition zones may indeed also be carried outwithout the presence of the marker, such a technology already beingknown in the state of the art. The applicants have in fact jointly fileda patent application PCT/FR00/02703 under French priority of 30 Sep.1999 on such a reading technique, which uses a photothermal method.

Among the direct methods for detection of hybridization, it is possibleto distinguish in particular the detection of the variation in mass, ofthe variation in thickness and of the variation in index. Photothermalmethods are also known which are described in the document by S. E.Bialkowski, vol. 137, under the title: “Photothermal spectrocopy methodsfor chemical analysis” taken from Chemical analysis: a series ofmonographs on analytical chemistry and its application, Wiley. Finally,the document U.S. Pat. No. 4,299,494 describes a technique forphotothermal deflection.

Thus, the invention finds applications in the fields of biological andchemical analysis.

In general, the reading of the molecular or immunological diagnosismentioned above has the main disadvantage of being limited byfalse-positives, that is to say hybrids or complexes which form whenthey should not have hybridized or formed a complex, and/orfalse-negatives, that is to say hybrids or complexes which do not formwhen they should have hybridized or formed a complex. This presence offalse-positives or of false-negatives causes another disadvantage, whichis the consequence of the first disadvantage mentioned above; themolecular diagnosis or the immunological assays lack sensitivity (powerto identify the hybrid or the complex sought when it is present in asmall quantity in a biological sample to be tested) and/or specificity(power to detect the hybrid or the complex sought in the biologicalsample to be tested containing other hybrids or complexes). This cancause diagnostic errors which are not acceptable for patients,practitioners and the companies manufacturing such tests.

The present invention proposes to solve all the disadvantages of theprior art mentioned above by providing a method of reading which islittle or not influenced by the presence of false-positives and whichtherefore very substantially improves the sensitivity and specificity ofdiagnostic tests.

To this effect, the present invention relates, according to a firstembodiment, to a method of reading on a support at least one biologicalreaction, which consists in:

-   -   bringing the support into contact with at least a first liquid        sample containing at least one group of biological recognition        molecules, which are identical to each other, so as to attach        these recognition molecules to the support, preferably at the        level of a recognition zone,    -   bringing the functionalized support into contact with at least a        second liquid sample containing at least one labeled target        biological molecule so as to attach this or these target        molecule(s) to said recognition molecule(s),    -   producing a first image of said support after this attachment of        the labeled target molecule(s),    -   treating the support under physicochemical conditions allowing        the separation of each target molecule with respect to a        recognition molecule to which it is specifically attached,    -   producing a second image of said support after this separation,        and    -   analyzing the two images in order to determine the specific        attachments between the recognition and target molecule(s).

According to a second embodiment, the present invention relates to amethod of reading on a support at least one biological reaction, whichconsists in:

-   -   bringing at least a first liquid sample containing at least one        group of biological recognition molecules, which are identical        to each other, into contact with at least a second liquid sample        containing at least one labeled target biological molecule so as        to attach this or these target molecule(s) to said recognition        molecule(s),    -   bringing the support into contact with the mixture of the at        least two first and second liquid samples containing at least        the group of biological recognition molecules optionally        complexed with at least one target molecule so as to attach this        or these recognition molecules or complex(es) to the support,        preferably at the level of a recognition zone,    -   producing a first image of said support after this attachment of        the labeled target molecule(s),    -   treating the support under physicochemical conditions allowing        the separation of each target molecule with respect to a        recognition molecule to which it is specifically attached,    -   producing a second image of said support after this separation,        and    -   analyzing the two images in order to determine the specific        attachments between the recognition and target molecule(s).

In the preceding two cases, the method may additionally comprise thefollowing additional subsequent steps:

-   -   bringing the functionalized and treated support, after specific        separation, into contact with the second liquid sample or with        another liquid sample so as to again attach at least one labeled        target biological molecule to:        -   the biological recognition molecule(s) from which it or they            had been separated, and/or        -   any other recognition molecule(s) derived from the same            recognition zone,    -   producing a third image of said support after this new        attachment of target molecule(s),    -   analyzing the third image with respect to the preceding analysis        of the two first images in order to confirm the specific        attachments between the recognition and target molecule(s).

According to a third embodiment, the present invention relates to amethod of reading on a support at least one biological reaction, whichconsists in:

-   -   bringing the support into contact with at least a first liquid        sample containing at least one group of biological recognition        molecules, which are identical to each other, so as to attach        these identical recognition molecules to the support, preferably        at the level of a recognition zone,    -   bringing the functionalized support into contact with at least a        second liquid sample containing at least one biological target        molecule so as to attach this or these target molecule(s) to        said recognition molecule(s),    -   bringing the functionalized and treated support into contact        with at least a third liquid sample containing at least one        labeled detection molecule so as to attach this or these        detection molecule(s) to said target molecule(s) attached to the        recognition molecule(s),    -   producing a first image of said support after this attachment of        the labeled detection molecule(s),    -   treating the support under physicochemical conditions allowing        the separation of each detection molecule with respect to a        target molecule to which it is specifically attached,    -   producing a second image of said support after this separation,        and    -   analyzing the two images in order to determine the specific        attachments between the target and detection molecule(s).

According to a fourth embodiment, the present invention relates to amethod of reading on a support at least one biological reaction, whichconsists in:

-   -   bringing at least a first liquid sample containing at least one        group of biological recognition molecules, which are identical        to each other, into contact with at least a second liquid sample        containing at least one biological target molecule so as to        attach this or these target molecule(s) to said recognition        molecule(s),    -   bringing the support into contact with the mixture of the at        least two first and second liquid samples containing at least        one group of identical biological recognition molecules, which        recognition molecule(s) is (are) optionally complexed with at        least one target molecule, so as to attach this or these        recognition molecule(s) and/or complex(es) to the support,        preferably at the level of a recognition zone,    -   bringing the functionalized and treated support into contact        with at least a third liquid sample containing at least one        labeled detection molecule so as to attach this or these        detection molecule(s) to said target molecule(s) attached to the        recognition molecule(s),    -   producing a first image of said support after this attachment of        the labeled detection molecule(s),    -   treating the support under physicochemical conditions allowing        the separation of each detection molecule with respect to a        target molecule to which it is specifically attached,    -   producing a second image of said support after this separation,        and    -   analyzing the two images in order to determine the specific        attachments between the target and detection molecule(s).

According to a fifth embodiment, the present invention relates to amethod of reading on a support at least one biological reaction, whichconsists in:

-   -   bringing at least a first liquid sample containing at least one        group of biological recognition molecules, which are identical        to each other, into contact with at least a second liquid sample        containing at least one biological target molecule so as to        attach this or these target molecule(s) to said recognition        molecule(s),    -   bringing the mixture of the two first and second liquid samples        into contact with at least a third liquid sample containing at        least one labeled detection molecule so as to attach this or        these detection molecule(s) to said target molecule(s) attached        to the recognition molecule(s),    -   bringing the support into contact with the mixture of the at        least three first, second and third liquid samples containing at        least the group of identical biological recognition molecules,        which recognition molecule(s) is (are) optionally complexed with        at least one target molecule which is itself optionally        complexed with at least one detection molecule so as to attach        this or these recognition molecules or complex(es) to the        support, preferably at the level of a recognition zone,    -   producing a first image of said support after this attachment of        the labeled detection molecule(s),    -   treating the support under physicochemical conditions allowing        the separation of each detection molecule with respect to a        target molecule to which it is specifically attached,    -   producing a second image of said support after this separation,        and    -   analyzing the two images in order to determine the specific        attachments between the target and detection molecule(s).

According to a sixth embodiment, the present invention relates to amethod of reading on a support at least one biological reaction, whichconsists in:

-   -   bringing at least a second liquid sample containing at least one        target biological molecule into contact with at least a third        liquid sample containing at least one biological detection        molecule, so as to attach this or these target molecule(s) to        the detection molecule(s),    -   bringing the mixture of the two second and third liquid samples        into contact with at least a first liquid sample containing at        least one group of recognition molecules, which are identical to        each other, so as to attach this or these recognition        molecule(s) to said target molecule(s), optionally attached to        said detection molecule(s),    -   bringing the support into contact with the mixture of the at        least three first, second and third liquid samples containing at        least the group of identical biological recognition molecules,        which recognition molecule(s) is (are) optionally complexed with        at least one target molecule which is itself optionally        complexed with at least one detection molecule so as to attach        this or these recognition molecules or complex(es) to the        support, preferably at the level of a recognition zone,    -   producing a first image of said support after this attachment of        the labeled detection molecule(s),    -   treating the support under physicochemical conditions allowing        the separation of each detection molecule with respect to a        target molecule to which it is specifically attached,    -   producing a second image of said support after this separation,        and    -   analyzing the two images in order to determine the specific        attachments between the target and detection molecule(s).

According to a seventh embodiment, the present invention relates to amethod of reading on a support at least one biological reaction, whichconsists in:

-   -   bringing the support into contact with at least a first liquid        sample containing at least one group of biological recognition        molecules, which are identical to each other, so as to attach        these identical recognition molecules to the support, preferably        at the level of a recognition zone,    -   bringing at least a second liquid sample containing at least one        target biological molecule into contact with at least a third        liquid sample containing at least one biological detection        molecule, so as to attach this or these target molecule(s) to        the detection molecule(s),    -   bringing the mixture of the at least two second and third liquid        samples containing at least one optionally complexed target        molecule into contact with a detection molecule so as to attach        this or these target molecule(s) to said recognition        molecule(s),    -   producing a first image of said support after this attachment of        the labeled detection molecule(s),    -   treating the support under physicochemical conditions allowing        the separation of each detection molecule with respect to a        target molecule to which it is specifically attached,    -   producing a second image of said support after this separation,        and    -   analyzing the two images in order to determine the specific        attachments between the target and detection molecule(s).

According to an eighth embodiment, the present invention relates to amethod of reading on a support at least one biological reaction, whichconsists in:

-   -   bringing at least a second liquid sample containing at least one        target biological molecule into contact with at least a third        liquid sample containing at least one biological detection        molecule, so as to attach this or these target molecule(s) to        the detection molecule(s),    -   bringing at least a first liquid sample containing at least one        group of biological recognition molecules, which are identical        to each other, into contact with the mixture of the at least two        second and third liquid samples containing at least one target        molecule optionally complexed with a detection molecule so as to        attach this or these target molecule(s) to said recognition        molecule(s),    -   bringing the support into contact with the mixture of the at        least three first, second and third liquid samples containing at        least one group of biological recognition molecules, which are        identical to each other, which recognition molecule(s) is (are)        optionally complexed with at least one target molecule, which is        itself optionally complexed with a detection molecule, so as to        attach these identical recognition molecules to the support,        preferably at the level of a recognition zone,    -   producing a first image of said support after this attachment of        the labeled detection molecule(s),    -   treating the support under physicochemical conditions allowing        the separation of each detection molecule with respect to a        target molecule to which it is specifically attached,    -   producing a second image of said support after this separation,        and    -   analyzing the two images in order to determine the specific        attachments between the target and detection molecule(s).

In the preceding five cases, the method may comprise the followingadditional subsequent steps:

-   -   bringing the functionalized and treated support, after specific        separation, into contact with the third liquid sample or with        another liquid sample, so as to again attach at least one        labeled detection molecule to:        -   the target molecule(s) from which it or they had been            separated, and/or        -   any other target molecule(s) (3) attached to one or more of            the recognition molecules (2) derived from the same            recognition zone,    -   producing a third image of said support after this new        attachment of detection molecule(s),    -   analyzing the third image with respect to the preceding analysis        of the two first images in order to confirm the specific        attachments between the target and detection molecule(s).

According to a first variant embodiment of the invention, at least onewashing is carried out after attachment:

-   -   of the recognition molecules to the support, the recognition        molecules being optionally attached to labeled target molecules        and/or to nonlabeled target molecules, the latter being        optionally attached to detection molecules, and/or    -   of the labeled target molecules and/or of the nonlabeled target        molecules to the recognition molecules, which are themselves        attached beforehand to said support, the target molecules being        optionally attached to detection molecules, and/or    -   of the detection molecules to nonlabeled target molecules, the        latter being attached to recognition molecules, which are        themselves attached to the support.

According to a second variant embodiment of said invention, the supportconsists of magnetic particles, and the method comprises steps ofmagnetizing said magnetic particles, which are preferably put in place:

-   -   during any step(s) of physicochemical treatment and/or of        producing an image, as described above, and/or    -   before any washing step, as described above.

According to a third variant embodiment of the invention, the bringingof the support into contact with at least two different first liquidsamples, optionally mixed beforehand, allows the attachment of at leasttwo different recognition molecules to said support in at least twodistinct recognition zones.

Regardless of the variant, when it is desired to quantify at least twooptionally different target molecules, the detection molecule consistsof a molecule combined directly or indirectly with at least one marker,such as a nanoparticle, whose means, which produce the images, allowindividual visualization, despite the presence of other neighboringdetection molecules and the like.

In all cases, the physicochemical conditions allowing the separation ofthe labeled target molecules with respect to the recognition moleculesor of the detection molecules with respect to the target molecules maybe obtained by heating to a melting point.

According to a preferred embodiment, the labeled target molecules, therecognition molecules or the target molecules consist of or thedetection molecules comprise nucleic acids.

According to another preferred embodiment, the labeled target molecules,the recognition molecules or the target molecules consist of or thedetection molecules comprise antibodies and/or antigens.

Whatever the case, the labeling of the target molecules or of thedetection molecules may be carried out by:

-   -   particles which can be visualized by conventional optical        microscopy, fluorescence microscopy, dark field microscopy or        atomic force microscopy,    -   molecules which can be visualized by atomic force microscopy,    -   enzymes with precipitating product which produce a detectable        signal, for example by fluorescence, luminescence, such as        horseradish peroxidase, alkaline phosphatase, β-galactosidase or        glucose-6-phosphate dehydrogenase,    -   chromophores such as fluorescent or luminescent compounds,    -   electronic markers which can be detected by electron microscopy,    -   absorbent molecules which can be visualized by thermal lens        microscopy.

In the case where three successive images of the support are produced,the other liquid sample, in place either of the second or of the thirdliquid sample, for bringing into contact the functionalized and treatedsupport, after specific separation, comprises a different marker fromthat contained in said second or third liquid sample.

The present invention also relates to a hybrid or complex obtainedduring the implementation of the method disclosed above, which is boundto a support by a recognition molecule, the hybrid or complex consistingof the recognition molecule bound to a target molecule which is itselfbound to a detection molecule. In this case, the bond between thedetection molecule and the target molecule is easier to dissociate undersuitable physicochemical conditions than the bond between said targetmolecule and the recognition molecule, and the bond between the targetmolecule and the recognition molecule is easier to dissociate undersuitable physicochemical conditions than the bond between saidrecognition molecule and the support.

In this case, when the recognition, target and detection molecules arebased on nucleic acids, the pairing between the detection molecule andthe target molecule comprises fewer total paired bases and/or comprisesfewer paired Guanine-Cytosine bases than the bond between the targetmolecule and the recognition molecule. Preferably, the number of totalpaired bases between the detection molecule and the target molecule isbetween five and fifty, preferably between ten and twenty five and stillmore preferably between twelve and twenty, and the number of totalpaired bases between the target molecule and the recognition molecule isbetween ten and one hundred, preferably between fifteen and fifty andmore preferably still between twenty and thirty. Furthermore, theproportion of paired Guanine-Cytosine bases involved in the bindingbetween the detection molecule and the target molecule is less than orequal to 50%, preferably less than or equal to 45%, and the proportionof paired Guanine-Cytosine bases involved in the binding between thetarget molecule and the recognition molecule is greater than or equal to50%, preferably greater than or equal to 55%.

The present invention finally relates to a biochip consisting of aplurality of hybrids or complexes, as described above, attached to atleast one support, as mentioned above.

On this biochip, the structurally identical hybrids or complexes aregrouped together to form recognition zones.

The accompanying figures and examples are given by way of an explanatoryexample and do not imply any limitation. They will make it possible tobetter understand the invention.

FIG. 1 represents a step of the method of reading consisting in theintroduction of a second liquid containing target molecules into abiochip which carries, for its part, recognition molecules.

FIG. 2 represents a step of the method of reading consisting in awashing to remove the nonhybridized or noncomplexed target molecules,that is to say which are not attached within the biochip.

FIG. 3 represents a step of the method of reading consisting in theintroduction of a third liquid containing detection molecules into abiochip which carries both recognition molecules and target molecules.

FIG. 4 represents a step of the method of reading consisting in awashing to remove the nonhybridized or noncomplexed detection molecules,that is to say which are not attached within the biochip.

FIG. 5 represents a step of the method of reading consisting inproducing a first image which represents the hybrids or complexesdetected on the biochip after the steps represented in FIGS. 1 to 4above.

FIG. 6 represents a step of the method of reading consisting in anoriented cleavage allowing dissociation between the target molecules andthe detection molecules.

FIG. 7 represents a step of the method of reading consisting in theproduction of a second image which represents the hybrids or complexesdetected on the biochip after the step represented in FIG. 6 above.

FIG. 8 represents a step of the method of reading consisting in thereintroduction of the third liquid containing detection molecules into abiochip which carries both recognition molecules and target molecules.

FIG. 9 represents a step of the method of reading consisting in awashing to remove the detection molecules which have not become attachedwithin the biochip.

FIG. 10 represents a step of the method of reading consisting inproducing a third image which represents the hybrids or complexesdetected on the biochip after the step represented in FIGS. 8 and 9above.

Finally, FIG. 11 represents the last step of the method of readingaccording to the invention, this step consists in the simultaneousanalysis of the second and third images in order to obtain a fourthimage which is truly representative of the hybrids or complexes detectedon the biochip.

The examples above will also make it possible to better understand theinvention.

EXAMPLE 1 Specific Dissociation of the Detection Molecules Combined withMagnetic Nanoparticles in Relation to Target Molecules

The model, chosen from HIV sequences, consists of:

-   -   a recognition molecule 2, biotinylated in 5′, to allow its        attachment to a support 1, corresponding to the sequence set        forth in SEQ ID NO: 1:5′-TCACTATTAT CTTGTATTAC TACTGCCCCT        TCACCTTTCC AGAGGAGCTT TGCTGGTCCT TTCCAAAGTG-3′ (length: 70        nucleotides),    -   a target molecule 3, corresponding to the sequence set forth in        SEQ ID NO: 2:

                              5′-ACAGCAGTACAAATGGCAGT ATTCATCCAC AATTTTAAAA GAAAAGGGGGGATTGGGGGG TACAGTGCAG GGGAAAGAAT AGTAGACATAATAGCAACAG ACATACAAAC TAAAGAATTA CAAAAACAAATTACAAAAAT TCAAAATTTT CGGGTTTATT ACAGGGACAG CAGAAATC CA   CTTTGGAAAG  GACCAGCAAA  GCTCCTCTGG AAAGGTGAAG   GGGCAGTAGT   AATACAAGAT  AATAGTGA CATAAAAGTAGT GCCAAGAAGA AAAGCAAAGA TCATTAGGGATTATGGAAAA CAGATGGCAG GTGATGATTG TGTGGCAAGTAGACAGGATG AGATTAGAAC ATGGAAAAGT TTAGTAAAACACCATATGTA TGTTTCAGGG AAAGCTAGGG GATGGTTTTATAGACATCAC TATGAAAGCC CTCATCC AAG   AATAAGTTCAGAAGTAAATC GAATTCCCGC GGCCATGGCG GCCGGGAGCATGCGACGTCG GGCCCAATTC GCCC-3′ (length: 514 nucleotides), and

-   -   a detection molecule 4, comprising an oligonucleotide        biotinylated at its 3′ end, corresponding to the sequence set        forth in SEQ ID NO: 3:5′-TTCTGAACTT ATTCTT-3′ (length: 16        nucleotides), and a marker.

In SEQ ID NO:2, part of the sequence (nucleotides 179-248 of SEQ IDNO:2) is represented in bold and underlined, this part corresponds tothe sequence complementary to SEQ ID NO:1 of the recognition molecule 2.Another part of the sequence (nucleotides 438-453 of SEQ ID NO:2) isrepresented solely underlined, this part corresponds to the sequencecomplementary to SEQ ID NO:3 of the detection molecule 4.

First Step: Immnobilization of the Recognition Molecules on the Support:

The support 1 used is a Corning glass slide (reference 0211) format 18mm×18 mm, silanized beforehand with 1% AMPMES(Amino-propyl-dimethyl-ethoxysilane).

Onto this support 1 is grafted neutravidin (Neutravidin biotin-bindingProtein Pierce reference 31000AH) via PDC (Phenylene diisothiocyanate)at a concentration of 1 mg/ml of PBS (Phosphate Buffer Saline) in theform of a deposit of 2 μl, that is 2 mm in diameter. This preliminarystep, not represented in the figures, allows subsequent attachment ofthe recognition molecules 2 to the support 1, by immobilization of thebiotinylated end of said recognition molecules 2 to the neutravidinpresent on said support 1.

After washing with a solution of 1% aqueous ammonia, 1 M NaCl, 1% BSA(Bovine Serum Albumin) and incubating in this same buffer for 10minutes, the support 1 is washed with water and then with TE buffer (10mM Tris at pH 8, 1 mM EDTA), 1 M NaCl, in order to remove theneutravidin not attached.

The support 1 onto which the neutravidin is attached is then incubatedfor 20 minutes at room temperature in the presence of recognitionmolecules 2, in solution in TE, 1 M NaCl, at a concentration of 5 μm.The support 1 is washed in a solution of 1% aqueous ammonia, 1 M NaCl,1% BSA by an incubation of 10 minutes. This support 1 is then immersedinto this same solution so as to saturate the totality of therecognition zones, for 20 minutes, washed with water and then with TE,so as to promote the removal of the recognition molecules 2 not attachedto the neutravidin.

The step of attachment of the recognition molecules 2 to the support 1via their biotinylated 5′ end is not represented in the figures, sincethe support 1, according to FIG. 1, is already functionalized to receivethe target molecules 3.

Second Step: Hybridization of the Target Molecules to the RecognitionMolecules:

After immobilization of the recognition molecules 2, the hybridizationthereof 2 with the target molecules 3 is carried out by incubation ofthe support in TE 1 M NaCl, 0.05% Triton X100 overnight at 35° C., whichcorresponds to FIG. 1. In the present case, 70 complementary base pairsare hybridized.

The support 1 is then taken up, washed in this same buffer for 15minutes at room temperature, as represented in FIG. 2, which makes itpossible to remove the target molecules 6 not hybridized.

It is interesting to note that according to one variant of theinvention, the recognition molecules 2 may be hybridized beforehand withthe target molecules 3 and the mixture is then brought into contactdirectly with the support 1.

Third Step: Labeling of the Oligonucleotides with Nanoparticles in Orderto Synthesize the Detection Molecules:

To detect the target molecules 3 hybridized with the recognitionmolecules 2, detection molecules 4 are labeled beforehand with magneticnanoparticles from Immunicon (Huntingdon Valley, USA; ref.: F3106).These nanoparticles have a diameter of 145 nm and are functionalizedwith streptavidin, generally 6000 to 20000 molecules of streptavidin areattached to a single nanoparticle, each streptavidin allowing theattachment of four biotins. These nanoparticles are incubated at aconcentration of 10⁹ particles/ml at 4° C. overnight in TE buffercontaining 1 M NaCl, 0.14 mg/ml salmon DNA, 0.05% Triton X100. Theoligonucleotide, forming the basis of the detection molecule 4,biotinylated at one of its ends, is then added to the solution ofnanoparticles, at the rate of one thousand detection oligonucleotidesper nanoparticle, and then incubated for 30 minutes at 35° C. Eachdetection oligonucleotide combined with a nanoparticle constitutes adetection molecule 4.

Fourth Step: 1st Hybridization of the Detection Molecules with theTarget Molecules:

Each step, represented in FIG. 3, consists in bringing the targetmolecules 3 hybridized with the recognition molecules 2 into contactwith the detection molecules 4. For this, the support 1, onto which areattached the target molecules 3 hybridized with the recognitionmolecules 2, is incubated with 1 ml of the solution of detectionmolecules 4, prepared according to the preceding step, at the rate of10⁹ nanoparticles/ml for 1 hour at room temperature. The hybridizationis performed on 16 complementary base pairs.

The support 1 is then washed in TE buffer containing 1 M NaCl, 0.05%Triton X100 for 15 minutes at room temperature so as to promote theremoval of the detection molecules not hybridized with the targetmolecules 3, as represented in FIG. 4.

It is advantageous to note that according to one variant of theinvention, the detection molecules 4 may be hybridized beforehand withthe target molecules 3 and the mixture is then directly brought intocontact with the support 1 to which the recognition molecules 2 areattached.

Fifth Step: Production of a 1st Image:

A first image 10 is produced by dark field microscopy (twenty (20) timesmagnification with a dark field lens) which makes it possible tovisualize nonfluorescent small sized nanoparticles, which corresponds toFIG. 5.

Sixth Step: Thermal Dissociation of the Target Molecules and of theDetection Molecules:

This dissociation step is represented in FIG. 6. The detection molecule4 possesses a melting point of 51° C. whereas the recognition molecule 2has a measured melting point of 90.5° C. A temperature of 60° C.constitutes a temperature greater than the melting point of thedetection molecule 4 and less than the melting point of the recognitionmolecule 2, allowing dissociation between the target molecule 3 and thedetection molecule 4. The dissociation of the detection molecules 4 isthus performed by treating the support 1 in a TE buffer containing 0.05%Triton X100 at 60° C. for 1 hour. Some detection molecules 4 remain atthe surface, but those which remain correspond to the detectionmolecules not specifically adsorbed at the surface.

This dissociation may also be obtained by incubating the support 1 inSodium Phosphate buffer at pH 5.5 at a concentration of 100 mM,containing 1 mM EDTA, 0.05% Triton X100 for 1 hour at 60° C. Thedissociation does not depend on the nature of the buffer used for thehybridization (Sodium Phosphate buffer at pH 5.5 and 100 mM, 1 M NaCl, 1mM EDTA, 0.05% Triton X100, or Sodium Phosphate buffer at pH 5.5 and 100mM, 0.1 M NaCl, 1 mM EDTA, 0.05% Triton X100). The conditions necessaryare the use of a low ionic strength, or even salt-free, buffer andheating for 1 hour at 60° C.

Seventh Step: Production of a Second Image:

There is produced by dark field microscopy a second image 11 which makesit possible to observe that most of the detection molecules 4 hybridizedbeforehand with the target molecules, which constituted true-positives,have disappeared. That is what is represented in FIG. 7.

This 2nd image 11 thus makes it possible to detect the detectionmolecules 4 which are only adsorbed at the surface or which were notproperly hybridized with the target molecules 3, that is to say withoutmismatch, which constitute false-positives.

By subtracting the detection spots present on the 1st image 10 fromthose of the 2nd image 11, it is possible to thus distinguish thepresence of true-positives and false-positives.

Eighth Step: 2nd Hybridization of the Detection Molecules with theTarget Molecules:

With the aim of further refining the results of the preceding steps, thesupport 1 is incubated a second time with the detection molecules 4under identical conditions to the first hybridization described above inthe fourth step, so as to reconstitute the labeling of the targetmolecules 3, already hybridized with the recognition molecules 2, withthe detection molecules 4. That is what is indeed represented in FIG. 8.

The support 1 is then washed in TE buffer containing 1 M NaCl, 0.05%Triton X100 for 15 minutes at room temperature, which corresponds toFIG. 9.

Ninth step: Production of a 3rd Image:

This 3rd image 12 makes it possible to refine the distinction madeduring the seventh step between the true-positives and thefalse-positives. Furthermore, the detection molecules 4 being specificfor the target molecules 3, this 3rd image 12 makes it possible toverify, as represented in FIG. 10, that said target molecules 3 arestill present on said recognition molecules 2, since the same intensityof the detection signal is obtained as that for the 1st image.Consequently, the target molecules 3 are not dissociated from therecognition molecules 2 during the treatment with TE containing TritonX100 at 60° C.: the dissociation is oriented.

It is also possible to carry out a final step corresponding to FIG. 11,where computer means can process the three images obtained 10, 11 and 12so as to precisely define the detection spots 18 really corresponding todetection molecules 4 hybridized with recognition molecule 2—targetmolecule 3 hybrids.

EXAMPLE 2 Oriented Dissociation of the Detection Molecules Combined withFluorescent Nanoparticles in Relation to Target Molecules

This example uses the biological model described in Example 1.

The two first steps described in Example 1, which correspondrespectively to the immobilization of the recognition molecules 2 on thesupport 1, and the hybridization of the target molecules 3 with therecognition molecules 2 are performed in a comparable manner in thisexample.

The third step, which corresponds to the labeling of the detectionmolecules on the other hand differs from Example 1 since thenanoparticles which were used for labeling the detection molecules 4 arefluorescent nanoparticles supplied by Molecular Probe (Eugene Oreg. USAref.: T8860) of 100 nm in diameter, already functionalized withneutravidin. The detection molecules 4 thus obtained are purified onMicrocon YM30 (Amicon MILLIPORE ref.: 42409).

The 1st hybridization of the detection molecules 4 with the targetmolecules is carried out as described in the fourth step of Example 1.

The counting of the detection spots is carried out by means of a 1stimage 10, obtained by fluorescence microscopy according to FIG. 5.

The dissociation step is carried out in TE buffer containing 0.05%Triton X100 at 60° C. for one hour, as represented in FIG. 7, asdescribed above in the sixth step of Example 1.

A second image 11 is produced by fluorescence microscopy. By subtractingthe intensity of fluorescence obtained in the first image from thatobtained in the second image, it is possible to deduce therefrom thefluorescence intensity due to the presence of false-positives. In acomparable manner to what is described in Example 1, a 2nd hybridizationof the detection molecules 4 with the target molecules 3 followed by athird image 12 makes it possible to check that a fluorescence intensityis indeed obtained which is comparable to that detected in the firstimage 10, demonstrating once again that the dissociation is indeedoriented.

The dissociation can therefore be performed with detection molecules 4labeled with nanoparticles of a different nature.

EXAMPLE 3 Oriented Dissociation of a Target Molecule Hybridized BetweenTwo Oligonucleotides Combined with Nanoparticles

The sequences of the recognition molecules 2, target molecules 3 anddetection molecules 4 used in this example are identical to thosedescribed in Example 1.

In this example, the invention is used with a double hybrid combiningthe target molecule 3 with:

-   -   the recognition molecule 2, itself 2 combined with a magnetic        particle, and    -   the detection molecule 4 labeled with a fluorescent        nanoparticle.

The use of a magnetic particle, which does not serve here as a marker,makes it possible to use a simple purification protocol by meremagnetization, and the use of a fluorescent particle makes it possibleto use fluorescence as the sensitive method for detecting and countingthe hybrids manufactured.

Thus, the recognition molecules 2 are combined with magnetic particles(Immunicon Streptavidine; Huntingdon Valley, USA, ref. F3106; diameter145 nm; concentration 10⁹ p/ml) while the detection molecules 4 arecombined with fluorescent nanoparticles (Molecular Probes Neutravidine;diameter 100 nm; concentration 10⁹ p/ml).

In a first stage, the target molecules 3 are hybridized with thefluorescent detection molecules 4. The target molecules 3(concentration: 10⁶ p/μl) are incubated at room temperature for 4 hourswith the detection molecules 4 (concentration: 1 nM) in TE buffercontaining 1 M NaCl and 0.05% Triton (final volume: 20 μl).

In a second stage, the target molecules 3 hybridized with the detectionmolecules 4 are incubated for 1 hour at room temperature with therecognition molecules 2 combined with the magnetic particles. The targetmolecules 3 are thus sandwiched between a magnetizable recognitionmolecule 2 and a fluorescent detection molecule 4.

In a third stage, the double hybrids are observed under a microscopewith epifluorescence illumination or with dark field illumination. Adevice with a permanent magnet, placed under the plate of themicroscope, makes it possible to apply a magnetic field of 300 Gauss.Under the action of this magnetic field, the magnetic particles assemblein the form of rods into which fluorescent particles may beincorporated. However, at this stage of the test, it cannot be concludedwhether these conjugates incorporate a target molecule or otherwise.

In a fourth stage, this uncertainty is removed at an orienteddissociation stage. For this, the buffer for suspending the particles isreplaced, under a magnetic confining field, by a low ionic strength TEbuffer containing 0.05% Triton X100, and the particles are heated at 70°C. for 30 minutes.

Immediately afterwards, a magnetic washing is carried out. Thesuspension obtained is examined under a microscope, still under amagnetic field of 300 Gauss. The combination of the ionic strength andthe temperature therefore made it possible to separate the targetmolecules 3 from the detection molecules 4. The level of dissociationdepends on the exact nature of the functionalizations of the two typesof nanoparticles and also on the length and the sequence of the DNAmolecules, and on the composition of the buffer.

This mode of application of oriented dissociation between a fluorescentparticle and a magnetic particle, or more precisely between a targetmolecule 3 and a detection molecule 4, can be carried out with othertypes of nonmagnetic or nonfluorescent particles, or with particleswhose functionalization is different, or with particles of differentsizes.

EXAMPLE 4 Oriented Dissociation of the Labeled Target Molecules inRelation to the Recognition Molecules

The main characteristic of this example is to directly use labeledtarget molecules without having recourse to detection molecules fordetecting the hybridization between the recognition molecules and thetarget molecules. The model consists of:

-   -   a recognition molecule, whose sequence is identical to that        described in Example 1    -   a target molecule, whose sequence is identical to that described        in Example 1, fragmented and labeled beforehand according to the        protocol described in patent application PCT/FR99/01469.

Using this method, fragments of about 50 nucleotides, labeled at their3′ end with a fluorescent marker, are obtained. One of these fragments,called detection fragment can hybridize by complementarity with therecognition molecule according to a protocol similar to that which isdescribed in Example 1.

Thus, the immobilization of the recognition molecules on the support iscarried out as described in the first stage of Example 1. Afterimmobilization of the recognition molecules, the hybridization of therecognition molecules with the labeled target molecules is carried outby incubating the support in TE containing 1 M NaCl.

The support 1 is then taken up, washed in this same buffer for 15minutes at room temperature. This washing step is very important sinceit makes it possible to remove the fragments obtained by this method,which are the detection fragments which are not hybridized, and thefragments which do not possess the region complementary to therecognition molecule.

A first image is produced by fluorescence microscopy, as described inExample 2, which makes it possible to obtain the level of fluorescencedue to the presence of true-positives, but also of false-positives.

The dissociation of the target molecules labeled with the recognitionmolecules is then carried out by incubating the support in a TE buffercontaining 0.05% Triton X100, at 95° C. for 1 h.

A second image is then produced which makes it possible to detect thedetection fragments which are only adsorbed at the surface and whichwere not properly hybridized with the recognition molecules, whichconstitute false-positives. As described in Example 1, by subtractingthe detection spots present on the 1st image from those of the 2ndimage, it is possible to distinguish between the presence oftrue-positives and false-positives.

Still with the aim of checking the results obtained in the precedingstages, the support to which the recognition molecules are attached isincubated a second time with the target molecules under conditionsidentical to the first recognition molecule—target moleculehybridization described above, and the support is washed in TE buffercontaining 1 M NaCl, 0.05% Triton X100 for 15 minutes at roomtemperature.

A 3rd image makes it possible to verify the distinction made between thetrue-positives and the false-positives. Furthermore, this 3rd imagemakes it possible to verify that said recognition molecules are stillpresent on the support since the same intensity of the detection signalis obtained as that for the 1st image. Consequently, the recognitionmolecules need not become dissociated from the support.

REFERENCES

-   1. Biochip support 5-   2. Recognition molecule-   3. Target molecule-   4. Detection molecule-   5. Biochip-   6. Nonhybridized or noncomplexed target molecule-   7. Target molecule hybridized or complexed elsewhere than on a    recognition molecule 2-   8. Nonhybridized or noncomplexed detection molecule-   9. Detection molecule adsorbed or complexed elsewhere than on a    target molecule 3-   10. First image-   11. Second image-   12. Third image-   13. Fourth image-   14. Detection spot corresponding to a detection molecule 4, 8 or 9-   15. Position of a detection spot which has disappeared,    corresponding to a detection molecule 4-   16. Detection spot corresponding to a detection molecule 9-   17. Detection spot which has just appeared, corresponding to a    detection molecule 9-   18. Detection spot corresponding to a detection molecule 4

1. A method of analyzing a support for at least one biological reaction,comprising: (a) bringing a support into contact with at least one firstliquid sample comprising at least one group of identical biologicalrecognition molecules so as to attach the recognition molecules to thesupport at the level of a recognition zone, such that the support isfunctionalized; b) bringing the functionalized support into contact withat least one second liquid sample comprising at least one labeled targetbiological molecule so as to attach the labeled target to therecognition molecules; (c) producing a first image of the support afterthe attachment of the labeled target showing detection spots; (d)separating the labeled target from the recognition molecules to whichthe labeled target is specifically attached; (e) producing a secondimage of the support after the separation in (d) showing detectionspots; and (f) analyzing the first and second images to determine thespecific attachments between the recognition molecules and labeledtarget by identifying true-positive and false-positive results bysubtracting the detection spots present in the second image from thedetection spots present in the first image.
 2. The method as claimed inclaim 1, wherein at least one washing step is carried out after theattachment of the recognition molecules to the support and/or after theattachment of the labeled target to the recognition molecules.
 3. Themethod as claimed in claim 2, wherein the support comprises magneticparticles.
 4. The method as claimed in claim 3, further comprisingmagnetizing the magnetic particles on the support during the step ofseparating the labeled target from the recognition molecules, duringimage production, or before any of the washing steps.
 5. The method asclaimed in claim 1, wherein the support is brought into contact with atleast two different first liquid samples, thereby allowing for theattachment of at least two different groups of recognition molecules tothe support in at least two distinct recognition zones.
 6. The method asclaimed in claim 1, further comprising: (g) bringing the support, afterthe separation in (d), into contact with the same at least one secondliquid sample so as to reattach the labeled target to the recognitionmolecules from which it had been separated, or into contact with adifferent second liquid sample so as to attach the labeled target toother recognition molecules from the same recognition zone; (h)producing a third image of the support after the attachment of thelabeled target in (g); and (i) analyzing the third image with respect tothe analysis of the first and second images to confirm the specificattachments between the recognition molecules and labeled target byidentifying true-positive and false-positive results.
 7. The method asclaimed in claim 6, wherein in the different second liquid sample, theat least one labeled target biological molecule has a different markerfrom that contained in the second liquid sample of step (b).
 8. Themethod as claimed in claim 1, wherein the separating of d) is obtainedby heating to a melting point.
 9. The method as claimed in claim 1,wherein the labeled target or recognition molecules comprise nucleicacids, antibodies, or antigens.
 10. The method as claimed in claim 1,wherein the labeled target is labeled for use with a method selectedfrom the group consisting of optical microscopy, fluorescencemicroscopy, dark field microscopy, atomic force microscopy, electronmicroscopy, and thermal lens microscopy.
 11. The method as claimed inclaim 1, wherein the labeled target is labeled using an enzyme having aprecipitating product which produces a detectable signal or using achromophore.
 12. The method as claimed in claim 11, wherein thedetectable signal is detected by fluorescence or luminescence.
 13. Themethod as claimed in claim 11, wherein the enzyme is selected from thegroup consisting of horseradish peroxidase, alkaline phosphatase,β-galactosidase, and glucose-6-phosphate dehydrogenase.
 14. The methodas claimed in claim 11, wherein the chromophore is a fluorescentcompound or a luminescent compound.
 15. A method of analyzing a supportfor at least one biological reaction, comprising: (a) mixing at leastone first liquid sample comprising at least one group of identicalbiological recognition molecules and at least one second liquid samplecomprising at least one labeled target biological molecule so as toattach the labeled target to the recognition molecules; (b) bringing asupport into contact with the mixture of the first and second liquidsamples so as to attach the recognition molecules to the support at thelevel of a recognition zone, such that the support is functionalized;(c) producing a first image of the support after the attachment of thelabeled target showing detection spots; (d) separating the labeledtarget from the recognition molecules to which the labeled target isspecifically attached; (e) producing a second image of the support afterthe separation in (d) showing detection spots; and (f) analyzing thefirst and second images to determine the specific attachments betweenthe recognition molecules and labeled target by identifyingtrue-positive and false-positive results by subtracting the detectionspots present in the second image from the detection spots present inthe first image.
 16. The method as claimed in claim 15, furthercomprising: (g) bringing the support, after the separation in (d), intocontact with the same at least one second liciuid sample so as toreattach the labeled target to the recognition molecules from which ithad been separated, or into contact with a different second liquidsample so as to attach the labeled target to other recognition moleculesfrom the same recognition zone; (h) producing a third image of thesupport after the attachment of the labeled target in (g); and (i)analyzing the third image with respect to the analysis of the first andsecond images to confirm the specific attachments between therecognition molecules and the labeled target by identifyingtrue-positive and false-positive results.
 17. The method as claimed inclaim 16, wherein in the different second liquid sample, the at leastone labeled target biological molecule has a different marker from thatcontained in the second liquid sample of step (b).
 18. A method ofanalyzing a support for at least one biological reaction, comprising:(a) bringing a support into contact with at least one first liquidsample comprising at least one group of identical biological recognitionmolecules so as to attach the recognition molecules to the support atthe level of a recognition zone, such that the support isfunctionalized; (b) bringing the functionalized support into contactwith at least one second liquid sample comprising at least onebiological target molecule so as to attach the taget to the recognitionmolecules; (c) bringing the functionalized support into contact with atleast one third liquid sample comprising at least one labeled detectionmolecule so as to attach the labeled detection molecule to the targetattached to the recognition molecules; (d) producing a first image ofthe support after the attachment of the labeled detection moleculeshowing detection spots; (e) separating the labeled detection moleculefrom the target to which the labeled detection molecule is specificallyattached; (f) producing a second image of the support after theseparation in (e) showing detection spots; and (g) analyzing the firstand second images to determine the specific attachments between thetarget and the labeled detection molecule by identifying true-positiveand false-positive results by subtracting the detection spots present inthe second image from the detection spots present in the first image.19. The method as claimed in claim 18, wherein at least one washing stepis carried out after the attachment of the recognition molecules to thesupport, after the attachment of the target to the recognitionmolecules, and/or after the attachment of the labeled detection moleculeto the target.
 20. The method as claimed in claim 19, wherein thesupport comprises magnetic particles.
 21. The method as claimed in claim20, further comprising magnetizing the magnetic particles on the supportduring step of separating the labeled detection molecule from thetarget, during image production, or before any of the washing steps. 22.The method as claimed in claim 18, wherein the support is brought intocontact with at least two different first liquid samples, therebyallowing for the attachment of at least two different groups ofrecognition molecules to the support in at least two distinctrecognition zones, wherein the different first liquid samples arecombined prior to or after contact with the support.
 23. The method asclaimed claim 22, wherein the support is brought into contact with atleast two different target molecules, thereby allowing for thequantification of said at least two different target molecules.
 24. Themethod as claimed in claim 23, wherein the labeled detection moleculecomprises a molecule combined with at least one marker which producesimages that allow individual visualization.
 25. The method as claimed inclaim 24, wherein the at least one marker is a nanoparticle.
 26. Themethod as claimed in claim 18, further comprising: (h) bringing thesupport, after separation in (e), into contact with the same at leastone third liquid sample so as to reattach the labeled detection moleculeand target from which it had been separated, or into contact with adifferent third liquid sample so as to attach the labeled detectionmolecule to any other target attached to the recognition molecules fromthe same recognition zone; (i) producing a third image of the supportafter the attachment of the labeled detection molecule in (h); and (j)analyzing the third image with respect to the analysis of the first andsecond images to confirm the specific attachments between the terget andthe labeled detection molecule by identifying true-positive andfalse-positive results.
 27. The method as claimed in claim 26, whereinin the different third liquid sample, the labeled labeled detectionmolecule has a different marker from that contained in the third liquidsample of step (c).
 28. The method as claimed in claim 18, wherein theseparating of e) is obtained by heating to a melting point.
 29. Themethod as claimed in claim 18, wherein the target, recognitionmolecules, or labeled detection molecule comprise nucleic acids,antibodies, or antigens.
 30. The method as claimed in claim 18, whereinthe labeled detection molecule is labeled for use with a method selectedfrom the group consisting of optical microscopy, fluorescencemicroscopy, dark field microscopy, atomic force microscopy, electronmicroscopy, and thermal lens microscopy.
 31. The method as claimed inclaim 18, wherein the labeled detection molecule is labeled using anenzyme having a precipitating product which produces a detectable signalor using a chromophore.
 32. The method as claimed in claim 31, whereinthe detectable signal is detected by fluorescence or luminescence. 33.The method as claimed in claim 31, wherein the enzyme is selected fromthe group consisting of horseradish peroxidase, alkaline phosphatase,β-galactosidase, and glucose-6-phosphate dehydrogenase.
 34. The methodas claimed in claim 31, wherein the chromophore is a fluorescentcompound or a luminescent compound.
 35. A method of analyzing a supportfor at least one biological reaction, comprising: (a) mixing at leastone first liquid sample comprising at least one group of identicalbiological recognition molecules and at least one second liquid samplecomprising at least one biological target molecule so as to attach thetarget to the recognition molecules; (b) bringing a support into contactwith the mixture of the first and second liquid samples so as to attachthe recognition molecules to the support at the level of a recognitionzone, such that the support is functionalized; (c) bringing thefunctionalized support into contact with at least one third liquidsample comprising at least one labeled detection molecule so as toattach the labeled detection molecule to the target attached to therecognition molecules; (d) producing a first image of the support afterthe attachment of the labeled detection molecule showing detectionspots; (e) separating the labeled detection molecule from the target towhich the labeled detection molecule is specifically attached; (f)producing a second image of the support after the separation in (e)showing detection spots; and (g) analyzing the first and second imagesto determine the specific attachments between the target and the labeleddetection molecule by identifying true-positive and false-positiveresults by subtracting the detection spots present in the second imagefrom the detection spots present in the first image.
 36. The method asclaimed in claim 35, further comprising: (h) bringing the support, afterseparation in (e), into contact with the same at least one third liquidsample so as to reattach the labeled detection molecule and the targetfrom which it had been separated, or into contact with a different thirdliquid sample so as to attach the labeled detection molecule to anyother target attached to the recognition molecules from the samerecognition zone; (i) producing a third image of the support after theattachment of the labeled detection molecule in (h); and (j) analyzingthe third image with respect to the analysis of the first and secondimages to confirm the specific attachments between the target and thelabeled detection molecule by identifying true-positive andfalse-positive results.
 37. The method as claimed in claim 36, whereinin the different third liquid sample, the labeled detection molecule hasa different marker from that contained in the third liquid sample ofstep (c).
 38. A method of analyzing a support for at least onebiological reaction, comprising: (a) mixing at least one first liquidsample comprising at least one group of identical biological recognitionmolecules and at least one second liquid sample comprising at least onebiological target molecule so as to attach the target to the recognitionmolecules; (b) adding at least one third liquid sample comprising atleast one labeled detection molecule to the mixture of the first andsecond liquid samples so as to attach the labeled detection molecule tothe target attached to the recognition molecules; (c) bringing a supportinto contact with the mixture of the first, second, and third liquidsamples so as to attach the recognition molecules to the support at thelevel of a recognition zone, such that the support is functionalized;(d) producing a first image of the support after the attachment of therecognition molecules showing detection spots; (e) separating thelabeled detection molecule from the target to which the labeleddetection molecule is specifically attached; (f) producing a secondimage of the support after the separation in (e) showing detectionspots; and (g) analyzing the first and second images to determine thespecific attachments between the target and the labeled detectionmolecule by identifying true-positive and false-positive results bysubtracting the detection spots present in the second image from thedetection spots present in the first image.
 39. The method as claimed inclaim 38, further comprising: (h) bringing the support, after separationin (e), into contact with the same at least one third liquid sample soas to reattach the labeled detection molecule and the target from whichit had been separated, or into contact with a different third liquidsample so as to attach the labeled detection molecule to any othertarget attached to the recognition molecules from the same recognitionzone; (i) producing a third image of the support after the attachment ofthe labeled detection molecule in (h); and (j) analyzing the third imagewith respect to the analysis of the first and second images to confirmthe specific attachments between the target and the labeled detectionmolecule by identifying true-positive and false-positive results. 40.The method as claimed in claim 39, wherein in the different third liquidsample, the labeled detection molecule has a different marker from thatcontained in the third liquid sample of step (c).
 41. A method ofanalyzing a support for at least one biological reaction, comprising:(a) mixing at least one second liquid sample comprising at least onetarget biological molecule and at least one third liquid samplecomprising at least one labeled detection molecule so as to attach thetarget to the labeled detection molecule; (b) adding at least one firstliquid sample comprising at least one group of identical recognitionmolecules to the mixture of the second and third liquid samples so as toattach the recognition molecules to the target; (c) bringing a supportinto contact with the mixture of the first, second, and third liquidsamples so as to attach the recognition molecules to the support at thelevel of a recognition zone, such that the support is functionalized;(d) producing a first image of the support after the attachment of thelabeled detection molecule showing detection spots; (e) separating thelabeled detection molecule from the target to which the labeleddetection molecule is specifically attached; (f) producing a secondimage of the support after the separation in (e) showing detectionspots; and (g) analyzing the first and second images to determine thespecific attachments between the target and the labeled detectionmolecule by identifying true-positive and false-positive results bysubtracting the detection spots present in the second image from thedetection spots present in the first image.
 42. The method as claimed inclaim 41, further comprising: (h) bringing the support, after separationin (e), into contact with the same at least one third liquid sample soas to reattach the labeled detection molecule and the target from whichit had been separated, or into contact with a different third liquidsample so as to attach the labeled detection molecule to any othertarget attached to the recognition molecules from the same recognitionzone; (i) producing a third image of the support after the attachment ofthe labeled detection molecule in (h); and (j) analyzing the third imagewith respect to the analysis of the first and second images to confirmthe specific attachments between the target and the labeled detectionmolecule by identifying true-positive and false-positive results. 43.The method as claimed in claim 42, wherein in the different third liquidsample, the labeled detection molecule has a different marker from thatcontained in the third liquid sample of step (a).
 44. A method ofanalyzing a support for at least one biological reaction, comprising:(a) bringing a support into contact with at least one first liquidsample comprising at least one group of identical biological recognitionmolecules so as to attach the recognition molecules to the support atthe level of a recognition zone, such that the support isfunctionalized; (b) mixing at least one second liquid sample comprisingat least one target biological molecule and at least one third liquidsample comprising at least one labeled detection molecule so as toattach the target to the labeled detection molecule; (c) bringing themixture of the second and third liquid samples into contact with thesupport so as to attach the target to the recognition molecules; (d)producing a first image of the support after the attachment of thelabeled detection molecule showing detection spots; (e) separating thelabeled detection molecule from the target to which the labeleddetection molecule is specifically attached; (f) producing a secondimage of the support after the separation in (e) showing detectionspots; and (g) analyzing the first and second images to determine thespecific attachments between the target and the labeled detectionmolecule by identifying true-positive and false-positive results bysubtracting the detection spots present in the second image from thedetection spots present in the first image.
 45. The method as claimed inclaim 44, further comprising: (h) bringing the support, after separationin (e), into contact with the same at least one third liquid sample soas to reattach the labeled detection molecule and the target from whichit had been separated, or into contact with a different third liquidsample so as to attach the labeled detection molecule to any othertarget attached to the recognition molecules from the same recognitionzone; (i) producing a third image of the support after the attachment ofthe labeled detection molecule in (h); and (j) analyzing the third imagewith respect to the analysis of the first and second images to confirmthe specific attachments between the target and the labeled detectionmolecule by identifying true-positive and false-positive results. 46.The method as claimed in claim 45, wherein in the different third liquidsample, the labeled detection molecule has a different marker from thatcontained in the third liquid sample of step (b).